10,11-epoxides of pga&#39; 2 &#39;and its 15-acetyl and -silyl derivatives

ABSTRACT

Novel methods are disclosed for transforming PGA2 and 15 Beta PGA2 and their acetates, methyl esters, and acetate methyl esters to various prostanoic acids and esters of the PGE2 and PGF2 series. Some of the latter are novel and are useful for the same pharmacological purposes as PGE2 and PGF2 .

United States Patent Pike et al.

[451 Sept. 9, 1975 10,1 l-EPOXIDES OF PGA AND ITS IS-ACETYL AND -SILYL DERIVATIVES Inventors: John E. Pike; William P. Schneider,

both of Kalamazoo, Mich.

Assignee: The Upjohn Company, Kalamazoo,

Mich.

Filed: Oct. 3, 1973 Appl. No.: 403,095

Related US. Application Data Division of Ser. No. 159,478, July 2, 1971, Pat. No. 3,772,350, which is a continuation-in-part of Ser. No. 71,390, Sept. 11, 1970, abandoned.

US. Cl. 260/348 C; 260/348 C Int. Cl. C07D 303/40 Field of Search 260/348 C [56] References Cited UNITED STATES PATENTS 3,736,336 5/1973 Strike et :al 260/348 C Primary Examiner--Norma S. Milestone Attorney, Agent, or Firm-Morris L. Nielsen [57] ABSTRACT 10 Claims, No Drawings 10,1 l-EPOXIDES OF PGA AND ITS IS-ACETYL AND -SILYL DERIVATIVES cRoss REFERENCE TO RELATED APPLICATIONS This is a division, of application Ser. No. 159,478, filed July 2, 1971, now issued as U.S. Pat. No. 3,772,350 which in turn is a continuation-in-part of our co-pending application Ser. No. 71,390, filed Sept. 1 l, 1970 and now abandoned. A

DESCRIPTION OF THE INVENTION This invention relates to novel compositions of matter, to novel methods for preparing them, and to novel intermediates used in those methods. This invention also relates to novel methods for preparing known compounds, and to novel intermediates used in those methods.

In particular, the several aspects of this invention relate to derivatives of prostanoic acid which has the following structure and numbering:

Some of the derivatives of prostanoic acid are known as prostaglandins. One of those, prostaglandin E (PGE has the following formula:

Anothen'prostaglandin F a (PGF a has the formula:

Still another, prostaglandin F 3 (PGF B has the formula:

In Formulas I to IV and in the formulas recited hereinafter in the specification and claims, broken line athydroxy at C-l5 is known as R or epi (beta), and is shown when necessary by attachment of said side-chain hydroxy to C-l5 with a heavy solid line and hydrogen with a dotted line, thus H OH.

The prostaglandin corresponding 'to PGE (Formula II) but with the R or epi configuration at C- l 5 will be designated l5,8-PGE See Nature, 212, 38 (1966) for discussion of the stereochemistry of the prostaglandins.

These conventions regarding formulas, names, and symbols for derivatives of prostanoic acid apply to the formulas, names, and symbols given hereinafter in the specification and claims. When reference is made hereinafter to the compounds of Formulas II to IV, by the symbols PGE PGFZa, 0r PGF or to the methyl esters of any of those, 15(8) configuration will be intended and by established custom, 8" or alpha will not be mentioned in the name or symbol. For all of the other compounds recited hereinafter, the configuration at C-l5 will be identified in the name as 1513 whenever the-l5(R) configuration is intended.

Molecules of the known prostaglandins each have several centers of asymmetry, and can exist in racemic (optically inactivelform and in either of the two enantiomeric (optically active) forms, i.e., the dextrorotatory and levorotatory forms. As drawn, Formulas "to IV each represent the particular optically active form of the prostaglandin which is obtained from certain mammalian tissues, for example, sheep vesicular glands, swine lung, or human seminal plasma, or by carbonyl and/or double bond reduction of a prostaglandin so obtained. See, for example,'Bergstrom et al., Pharmacol. Rev. 20, 1 1968) and references cited therein.

The several aspects of this invention relate to novel methods for preparing PGE PGFZQ, and PGE their acetates and methyl est-ers,and the 15 B-epimers of those compounds, to novel intermediates used in those methods, to novel methods used to make those intermediates, and to certain novel and pharmacologically useful analogs of PGE PGF and PGF The novel and 'pharmacologically useful PGE PGF and PGF analogs: of this invention have the formulas:

Vll

In Formulas V, VI, and VII, R, is hydrogen, alkyl of one to 8 carbon atoms, inclusive, cycloalkyl of 3 to 10 carbon atoms, inclusive, aralkyl of 7 to 12 carbon atoms, inclusive, phenyl, or phenyl substituted with one to 3 chloro or alkyl of one to 4 carbon atoms, inclusive. Also encompassed by Formulas V, VI, and VII are pharmacologically acceptable salts when R, is hydrogen. In Formulas V and VII,

HOH

In Formula V, B is In Formula VI, indicates attachment to the ring in alpha or beta configuration.

It will be observed that each of the novel compounds of Formulas V and VI has a hydroxy group attached to the l l-position in beta configuration. In PGE PGFzmand PGF and in the compounds of Formula VII, the hydroxy at C-1 1 is attached in alpha configuration.

With regard to Formulas V, VI, and VII, examples of alkyl of one to 8 carbon atoms, inclusive, are methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, and isomeric forms thereof. Examples of cycloalkyl of 3 to carbon atoms, inclusive, which includes alkylsubstituted cycloalkyl, are cyclopropyl, Z-methylcyclopropyl, 2,Z-dimethylcyclopropyl, 2,3-diethylcyclopropyl, Z-butylcyclopropyl, cyclobutyl, 2- methylcyclobutyl, 3-propylcyclobutyl, 2,3,4-triethylcyclobutyl, cyclopentyl, 2,Z-dimethylcyclopentyl, 3-pentylcyclopentyl, 3-tert-butylcyclopentyl, cyclohexyl, 4-tert-butylcyclohexyl, 3-isopropylcyclohexyl, 2,2-dimethylcyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, and cyclodecyl. Examples of aralkyl of 7 to l2 carbon atoms, inclusive, are benzyl, phenethyl, lphenylethyl, Z-phcnylpropyl, 4-phenylbutyl, 3- phenylbutyl, 2-( l-naphthylethyl), and l-( 2- naphthylmethyl). Examples of phenyl substituted by one to 3 chloro or alkyl of one to 4 carbon atoms, inclu sive, are p-chlorophcnyl, m-chlorophenyl, ochlorophenyl, 2,4-dichlorophe nyl, 2,4,6- trichlorophenyl, p-tolyl, m-tolyl, o-tolyl, p-ethylphenyl, p-tert-butylphenyl, 2,5-dimethylphenyl, 4-chloro-2- methylphenyl, and 2,4-dichloro-3-methylphenyl.

The known prostanoic acid derivatives, PGE- PGF2a, and PGF and their esters and pharmacologically acceptable salts are extremely potentin causing various biological responses. For that reason, these compounds are useful for pharmacological purposes.

See, for example, Bergstrom et al., cited above, and references cited therein. A few of those biological re sponses are systemic arterial blood pressure lowering in the case of the PGE and PGE compounds as measured, for example, in anesthetized (pentobarbital sodium) pentolinium-treated rats with indwelling aortic and right heart cannula; pressor activity, similarly measured, for the PGF or compounds; stimulation of smooth muscle as shown, for example, by tests on strips of guinea pig ileum, rabbit duodenum, or gerbil colon; potentiation of other smooth muscle stimulants; antilipolytic activity as shown by antagonism of epinephrine-induced mobilization of free fatty acids or inhibition of the spontaneous release of glycerol from isolated rat fat pads; inhibition of gastric secretion in the case of the PGE compounds as shown in dogs with secretion stimulated by food or histamine infusion; activity on the central nervous system; controlling spasm and facilitating breathing in asthmatic conditions; decrease of blood platelet adhesiveness as shown by platelet-toglass adhesiveness, and inhibition of blood platelet aggregation and thrombus formation induced by various physical stimuli, e.g., arterial injury, and various biochemical stimuli, e.g., ADP, ATP, serotonin, thrombin, and collagen; and in the caseof the PGE compounds, stimulation of epidermal proliferation and keratinization as shown when applied in culture to embryonic chick and rat skin segments.

Because of these biological responses, these known prostaglandins are useful to study, prevent, control, or alleviate a wide variety of diseases and undesirable physiological conditions in birds and mammals, including humans, useful domestic animals, pets, and zoological specimens, and in laboratory animals, for example, mice, rats, rabbits, and monkeys.

For example, these compounds, and especially the PGE compounds, are useful in mammals, including man, as nasal decongestants. For this purpose, the compounds are used in a dose range of about l0,u.g. to about 10 mg. per ml. of a pharmacologically suitable liquid vehicle or as an aerosol spray, both for topical application.

The PGE and PGFa compounds are useful in the treatment of asthma. For example, these compounds are useful as bronchodilators or as inhibitors of mediators, such as SRS-A and Histamine which are released from cells activated by an antigen-antibody complex. Thus, these compounds control spasm and facilitate breathing in conditions such as bronchial asthma, bronchitis, bronchiectasis, pneumonia and emphysema. For these purposes, these compounds are administered in a variety of dosage forms, e.g., orally in the form of tablets, capsules, or liquids; rectally in the form of tablets, capsules, or liquids; rectally in the form of supposito ries; parenterally, subcutaneously, or intramuscularly, with intravenous administration being preferred in emergency situations; by inhalation in the form of aerosols or solutions for nebulizers; or by insufflation in the form of powder. Doses in the range of about 0.01 to 5 mg. per kg. of body weight are used 1 to 4 times a day, the exact dose depending on the age, weight, and condition of the patient and on the frequency and route of administration. For the above use these prostaglandins can be combined advantageously with other antiasthmatic agents, such as sympathomimetics (isoproterenol, phenylephrine, ephedrine, etc); xanthine derivatives (theophylline and aminophyllin); and corticosteroids (ACTH and predinisolone). Regarding use of these compounds see South African Patent No. 681,055.

The PGE compounds are useful in mammals, including man and certain useful animals, e.g., dogs and pigs, to reduce and control excessive gastric secretion, thereby reducing or avoiding gastrointestinal ulcer formation, and accelerating the healing of such ulcers already present in the gastrointestinal tract. For this purpose, the compounds are injected or infused intravenously, subcutaneously, or intramuscularly in an infusion dose range about 0.1 ,ug. to about 500 pg. per kg. of body weight per minute, or in a total daily dose by injection or infusion in the range about 0.1 to about 20 mg. per kg. of body weight per day, the exact dose depending on the age, weight, and condition of the patient or animal, and on the frequency and route of ad ministration. I

The PGEz, PGFzm and PGF compounds are useful whenever it is desired to inhibit platelet aggregation, to reduce the adhesive character of platelets,'and to remove or prevent the formation of thrombi in mammals, including man, rabbits, and rats. For example, these compounds are useful in the treatment and prevention of myocardial infarcts, to treat and prevent postoperative thrombosis, to promote patency of vascular grafts following surgery, and to treat conditions such as atherosclerosis, arteriosclerosis, blood clotting defects due to lipemia, and other clinical conditions in which the underlying etiology is associates with lipid imbalance or hyperlipidemia. For these purposes, these compounds are administered systemically, e.g., intravenously, subcutaneously, intramuscularly, and in the form of sterile implants for prolonged action. For rapid response, especially in emergency situations, the intravenous route of administration is preferred. Doses in the range about 0.005 to about 20 mg. per kg. of body weight per day are used, the exact dose depending on the age, weight, and condition of the patient or animal, and on the frequency and route of administration.

The PGE P6P and PGF compounds are especially useful as additives to blood, blood products, blood substitutes, and other fluids which are used in artificial extracorporeal circulation and perfusion of iso lated body portions, e.g., limbs and organs, whether attached to the original body, detached and being preserved or prepared for transplant, or attached to a new body. During these circulations and perfusions, aggregated platelets tend to block the blood vessels and portions of the circulation apparatus. This blocking is avoided by the presence of these compounds. For this purpose, the compound is added gradually or in single or multiple portions to the circulating blood, to the blood of the donor animal, to the perfused body portion, attached or detached, to the recipient, or to two or all of those at a total steady state dose of about 0.00 1 to 10 mg. per liter of.circulating fluid. It is especially useful to use these compounds in laboratory animals, e.g., cats, dogs, rabbits, monkeys, and rats, for these purposes in order to develop new methods and techniques for organ and limb transplants.

The PGE compounds are extremely potent in causing stimulation of smooth muscle, and are also highly active in potentiating other known smooth muscle stimulators, for example, oxytocic agents, e.g., oxytocin, and the various ergot alkaloids including derivatives and analogs thereof. Therefore PGEg, for example, is useful in place of or in combination with less than usual amounts of these known smooth muscle stimulators, for example, to relieve the symptoms of paralytic ileus, or to control or prevent atonic uterine bleeding after abortion or delivery, to aid in expulsion of the placenta, and during the puerperium. For the latter purpose, the PGE compound is administered by intravenous infusion immediately after abortion or delivery at a dose in the range about 0.01 to about 50 ,ug. per kg. of body weight per minute until the desired effect is obtained. Subsequent doses are given by intravenous, subcutaneous, or intramuscular injection or infusion during puerperium in the range 0.01 to 2 mg. per kg. of body administered by intravenous infusion at the rate about 0.01 to about 50 ug. per kg. of body weight per minute, or in singleor multiple doses of about 25 to 500 ,ug. per kg. of body weight total per day.

The PGE PGA and PGFZ compounds also increase the flow of blood in the mammalian kidney, therein increasing volume and electrolyte content of the urine. Therefore, these compounds are useful in managing cases of renal disfunction, especially those involving blockage of the' renal vascular bed. Illustratively, the compounds are useful to alleviate and correctcases of edema resulting, for example, from massive surface burns, and in the management of shock; For these purposes, the compounds are preferably first administered by intravenous injection at a dose in the range 10 to 1000 pg per kg. of body weight or by intravenous infusion at a dose in the range 0. l to 20 pg. per kg. of body weight per minute until the desired effect is obtained. Subsequent doses are given by intravenous, intramuscular, or subcutaneous injection or infusion in the range'0.05 to 2 mg. per kg. of body weight per day.

The PGE PGF and PGF compounds are useful in place of oxytocin to induce labor-in pregnant female animals, including man, cows, sheep, and pigs, at or near term, or in pregnantanimals with intrauterine death of the fetus from about 20 weeks to term. For this purpose, the compound isin fused intravenously at a dose 0.01 to 50 pg. per kg. ofbody weight per minute until or near the termination of the second stage of labor, i.e.; expulsion of the fetus! These compounds are especially useful when the female is one or more weeks post-mature and natural labor has not started, or 12 to 60 hours after the membranes have ruptured and natural labor has not yet started.

The PGF a PGR and PGE compounds are useful for controlling the reproductive cycle in ovula ting female mammals, including humans and animals such as monkeys, rats, rabbits, dogs, cattle, and the like. For that purpose, PGE or PGF a for example, is administered systemically, e.g., intravenously, *subcuaneously, and intravaginally, at a dose level in the range 0.00] mg. to about 20 mg. per kg. of body weight of the female mammal, advantageously during a span of time starting approximately at the time of ovulation and ending approximately at the next expected time of menses or just prior to that timenAdditionally, expulsion of an embryoor fetus (abortion) is accomplished by similar administrataion of the compound during the first third of the normal mammalian gestation period.

As mentioned above, the P'GE compounds are potent antagonists of epinephrine-induced mobilization of free fatty acids. For this reason, this compound is useful in experimental medicine for both in vitro and in vivo studies in mammals, including man, rabbits, and rats, intended to lead to the understanding, prevention, symptom alleviation, and cure of diseases involving abnormal lipid'mobilization and high free fatty acid levels, e.g., diabetes mellitus, vascular diseases, and hyperthyroidism.

The novel Formula-V, -VI, and -VII PGE F'GF a and PGF analogs of this invention each cause the biological responses described above for PGE PGF a, and PGFQ respectively, and each of these novel compounds is accordingly useful for the abovedescribed corresponding purposes, and is used for those purposes in the same manner as described above.

PGEZ, PGFZQ, and PGF and their esters and pharmacologically acceptable salts are all potent in causing multiple biological responses even at low doses. For example, PGE is extremely potent in causing vasodepression and smooth muscle stimulation, and is also potent as an antilipolytic agent. Moreover, for many applications, these known prostaglandins have an inconveniently short duration of biological activity. In striking contrast, the novel analogs of Formulas V, VI, and VII are substantially more specific with regard to potency in causing prostaglandin-like biological responses, and have a substantially longer duration of biological activity. Therefore, each of these novel prostaglandin analogs is surprisingly and unexpectedly more useful than one of the corresponding abovementioned known prostaglandins for at least one of the pharmacological purposes because it has a different and narrower spectrum of biological activity than the known prostaglandins, and therefore is more specific in its activity and causes smaller and fewer undesired side effects than when the known prostaglandin is used for the same purpose. Moreover, because of its prolonged activity, fewer and smaller doses of the novel prostaglandin analog can frequently be used to attain the desired result.

The novel Formula-V, -VI, and -VII prostaglandin analogs are used as described above in free acid form in ester form, or in pharmacologically acceptable salt form. When the ester form is used, the alkyl esters are preferred, especially the alkyl esters wherein the alkyl moiety contains one to 4 carbon atoms, inclusive. Of those alkyl, methyl and ethyl are especially preferred for optimum absorption of the compound by the body or experimental animal system.

Pharmacologically acceptable salts of these prostaglandin analogs useful for the purposes described above are those with pharmacologically acceptable metal cations, ammonium, amine cations, or quaternary ammonium cations.

Especially preferred metal cations are those derived from the alkali metals, e.g., lithium, sodium and potassium, and .from the alkaline earth metals, e.g., magnesium and calcium, although cationic forms of other metals, e.g., aluminum, zinc and iron, are within the scope of this invention.

Pharmacologically acceptable amine cations are those derived from primary, secondary, or tertiary amines. Examples of suitable amines are methylamine, dimethylamine, trimethylamine, ethylamine, dibutylamine, triisopropylamine, N-methylhexylamine, decylamine, dodecylamine, allylamine, crotylamine, cyclopentylamine, dicyclohexylamine, benzylamine. dibenzylamine, a-phenylethylamine, ,B-phenylethylamine, ethylenedimaine, diethylenetriamine, and like aliphatic, cycloaliphatic, and araliphatic amines containing up to and including about 18 carbon atoms, as well as heterocyclic amines, e.g., piperidine, morpholine, pyrrolidine, piperazine, and lower-alkyl derivatives thereof, e. g. I -methylpiperidine, 4- ethylmorpholine, l-isopropylpyrrolidine, 2- methylpyrrolidine, l ,4-dimethylpiperazine, 2-

methylpiperidine, and the like, as well as amines containing water-solubilizing or hydrophilic groups, e.g.,

mono-, di-, and triethanolamine, ethyldiethanolamine, N-butylethanolamine, 2-amino-l-butanol, 2-amino-2- ethyl-1,3-propanediol, 2-amino-2-methyll -propanol, tris( hydroxymethyl )aminomethane, N- phenylethanolamine, N-( p-tert-amylphenyl )diethanolamine, galactamine, N-methylglucamine, N- methylglucosamine, ephedrine, phenylephrine, epinephrine, procaine, and the like.

Examples of suitable pharmacologically acceptable quaternary ammonium cations are tetramethylammonium, tetraethylammonium, benzyltrimethylammonium, phenyltriethylammonium, and the like.

As discussed above, these novel prostaglandin analogs are administered in vasious ways for various purposes; e.g., intravenously, intramuscularly, subcutaneously, orally, intravaginally, rectally, buccally sublingually, topically, and in the form of sterile implants for prolonged action.

For intravenous injection or infusion, sterile aqueous isotonic solutions are preferred. For that purpose, it is preferred because of increased water solubility to use the free acid form or the pharmacologically acceptable salt form. For subcutaneous or intramuscular injection, sterile solutions or suspensions of the acid, salt, or ester form in aqueous or non-aqueous media are used. Tablets, capsules, and liquid preparations such as syrups, elixirs, and simple solutions, with the usual pharmaceutical carriers are used for oral or sublingual administration. For rectal or vaginal administration, suppositories, tampons, ring devices, and preparations adapted to generate sprays or foams or to be used for lavage, all prepared as known in the art, are used. For tissue implants, a sterile tablet or silicone rubber capsule or other object containing or impregnated with the substance is used.

The novel compounds of Formulas V, VI, and VII wherein R is other than hydrogen, i.e., the esters wherein R, is alkyl of one to 8 carbon atoms, inclusive, cycloalkyl of 3 to 10 carbon atoms, inclusive, aralkyl of 7 to 12 carbon atoms, inclusive, phenyl, or phenyl substituted with one to 3 chloro or alkyl of one to 4 carbon atoms, inclusive, are prepared from the corresponding acids of Formulas V, VI, and VII, i.e., wherein R is hydrogen, by methods known in the art. For example, the alkyl, cycloalkyl, and aralkyl esters are prepared by interaction of said acids with the appropriate diazohydrocarbon. For example, when diazomethane is used, the methyl esters are produced. Similar use of diazoethane, diazobutane, l-diazo-2-ethylhexane, diazocyclohexane, and phenyldiazomethane, for example, gives the ethyl, butyl, Z-ethylhexyl, cyclohexyl, and benzyl esters, respectively.

Esterification with diazohydrocarbons is carried out by mixing a solution of the diazohydrocarbon in a suitable inert solvent, preferably diethyl ether, with the acid reactant, advantageously in the same or a different inert diluent. After the esterification reaction is complete, the solvent is removed by evaporation, and the ester purified if desired by conventional methods, preferably by chromatography. It is preferred that contact of the acid reactants with the diazohydrocarbon be no longer than necessary to effect the desired esterification, preferably about one to about ten minutes, to avoid undesired molecular changes. Diazohydrocarbons are known in the art or can be prepared by methods known in the art. See, for example, Organic Reactions, John Wiley & Sons, Inc., New York, N.Y., Vol. 8, pp. 389-394 (1954).

An alternative method for esterification of the carboxyl moiety of the novel PGF-type or PGE-type compounds of Formulas V, VI, and VII comprises transformation of the free acid to the corresponding silver salt, followed by interaction of that salt with an alkyl iodide. Examples of suitable iodides are methyl iodide, ethyl iodide, butyl iodide, isobutyl iodide, tert-butyl iodide, cyclopropyl iodide, cyclopentyl iodide, benzyl iodide, phenethyl iodide, and the like. The silver salts are prepared by conventional methods, for example, by dissolving the acid in cold dilute aqueous ammonia, evaporating the excess ammonia at reduced pressure, and then adding the stoichiometric amount of silver nitrate.

The phenyl and substituted phenyl esters of the Formula-V, VI, and -VII compounds are prepared by silyiating the acid to protect the hydroxy groups, for example, replacing each OI-I with -OSiCI-I Doing that may also change COOH to COOS-Si- -(CH A brief treatment of the silylated compound with water will change COO--Si-(CH;,) back to COOH. Procedures for this silylation are known in the art and are discussed hereinafter. Then, treatment of the silylated compound with oxalyl chloride gives the acid chloride which is reacted with phenol or the appropriate substituted phenol to give a silylated phenyl or substituted phenyl ester. Then the silyl groups, e.g., O-Si(CH;,) are changed back to OH by treatment with dilute acetic acid. Procedures for these transformations are known in the art.

The novel Formula-V, VI, and VII acids (R is hydrogen) are transformed to pharmacologically acceptable salts by neutralization with appropriate amounts of the corresponding inorganic or organic base, examples of which correspond to the cations and amines listed above. These transformations are carried out by a variety of procedures known in the art to be generally useful for the preparation of inorganic, i.e., metal or ammonium salts, amine acid addition salts, and quaternary ammonium salts. The choice of procedure depends in part upon the solubility characteristics of the particular salt to be prepared. In the case of the inorganic salts, it is usually suitable to dissolve the acid in water containing the stoichiometric amount of a hydroxide, carbonate, or bicarbonate corresponding to the inorganic salt desired. For example, such use of sodium hydroxide, sodium carbonate, or sodium bicarbonate gives a solution of the sodium salt of the prostanoic acid derivative. Evaporation of the water or addition of a watermiscible solvent of moderate polarity, for example, a lower alkanol or a lower alkanone, gives the solid inorganic salt if that form is desired.

To produce an amine salt, the acid is dissolved in a suitable solvent of either moderate or low polarity. Examples of the former are ethanol. acetone, and ethyl acetate. Examples of the latter are diethyl ether and benzene. At least a stoichiometric amount of the amine corresponding to the desired cation is then added to that solution. If the resulting salt does not precipitate, it is usually obtained in solid form by addition of a miscible diluent of low polarity or by evaporation. If the amine is relatively volatile, any excess can easily be removed by evaporation. It is preferred to use stoichiometric amounts of the less volatile amines.

Salts wherein the cation is quaternary ammonium are produced by mix the acid with the stoichiometric amount of the corresponding; quaternary ammonium hydroxide in water solution, followed by evaporation of the water.

The novel compounds of Formulas V, VI. and VII wherein R, is hydrogen or methyl, i.e., the free acids and the methyl esters, and also PGE PGF a and PGF B and the methyl esters of those are prepared by novel methods which are described hereinafter. For those methods, one of the following starting materials is used:

In Formulas VIIa, VIII, and IX, R is either hydrogen or methyl, and R is hydrogen or acetyl.

It will be observed that the compounds encompassed by Formula VIIa are also encompassed by VII. Thus, some Formula-VII compounds are useful both as intermediates and for pharmacological purposes.

These Formula-VlIa, VIII, and IX starting materials are all derivatives of prostanoic acid. The compounds of Formula VIII are known in the art or are available by methods known in the art. See, for example, Weinheimer et al., Tetrahedron Letters, No. 59, 5185 (1969); H.W. Youngken, .Ir. (ed), Food-Drugs from the Sea, Proc. Marine Technology Society, pp. 3l 1-3 14 1969). The Formula-VIII compound wherein R and R are both hydrogen is designated l5B-PGA alternatively l5(R)-PGA or l5-epi-PGA The other compounds encompassed by Formula VIII are designated 15,8-PGA acetate, l5/3-PGA methyl ester, and l5B-PGA acetate methyl ester.

The compounds of Formula VIIa are new in the art and methods for preparing them are described hereinafter. The Formula-VIIa compound wherein R and R are both hydrogen is designated lSfi-PGE alternatively l5(R)-PGE or l5-epi-PGE The other compounds encompassed by Formula VIIa are designated l5B-PGE IS-acetate, l5B-PGE methyl ester, and l5B-PGE acetate methyl ester.

The compounds of Formula 11X are known in the art. See, for example, British Specification 1,097,533. Novel methods for preparing these Formula-IX compounds are described hereinafter. The Formula-IX compound wherein R and R are hydrogen is designated PGA The other compounds encompassed by Formula IX are designated PGA acetate, PGA: methyl ester, and PGA acetate methyl ester.

All of the compounds of Formulas VIIa, VIII, and IX are obtained by extraction from a marine invertebrate. The compounds of Formula VIIa and VIII, i.e., the 158 compounds, are obtained from colonies of Plexaura Immomalla (Esper), I792, formla R. The compounds of Formula IX i.e., the 15(S) or'alpha compounds, are obtained from colonies of Plat-aura homomalla (Esper),

1792, forma S. s s s These Plexc ura homomalla forms are members of the subclass Octocorallia, order. Gorgona'cea, suborder Holaxonia, familyPlexauridae, genus Plex'aura. See, for example, Bayer, The Shallow-WaterOctocorallia of the West Indian Region, Martinus Nijhoff, The Hague (1961). Colonies of these Plxaura hbmomalla forms are abundant on the ocean reefs in the zone from the low-tide line to about 25 fathoms in the tropical and subtropical regions of the western part of theAtlantic Ocean, from Bermuda to the reefs of Brazil, including the eastern shore reefs of Florida the Caribbean island and mainland reefs, and the Gulf of Mexico island and mainland reefs. These colonies are bush-like or small tree-like in habit, and are readily identified for collec tion as Plexuura llmnomallu(Esper), 1792, by those of ordinary skill in this art. Forms R and S are distinguished by the methods described in Preparation 1 below.

The colonies of these two forms of Plexuura Immumallu are easily separated into an outer bark-like cortex and an inner wiry proteinaceous stem or skelton. Symbiotic algae or Zooxanthellae areialso present in the colonies. Weinheimer et al., cited above, disclose the occurrence of the Formula-V111. compounds wherein R and R are both hydrogen and wherein R is methyl and R is acetyl in the air dried cortex of Plaraurrz homomalla (Esper).

The choice of isolation or extraction method is determined by the particular Formula-Vlla, -Vlll, or -lX compound desired. Maximum yield of the Formula- Vlll or -lX diester is realized freezing whole or coarselycut or chopped fresh VIP/(QUINN! homomalla colonies within an hourand preferably sooner after the colonies are removed from the reef. For small scale collections, this freezing is done advantageously by contactingthe colonies or pieces with solid carbon dioxide. For larger scale collections, other suitable freezing methods are known to the art. The frozen colonies or colony pieces should be kept frozen, preferably below about 2() C until the extraction takes place.

The major component of fresh Plexuura hommiuillu (Esper), 1792, forma R is ISB-PGA acetate methyl ester, the Formula-V111 compound wherein R is methyl and R is acetyl. Relatively minor components are the hydroxy methyl ester, the acetate, and the hydroxy acid of Formula V111 and the l5B-lC-E compounds'encompassed by Formula Vlla. Of the latter, the 15,8-PGE acetate methyl ester (R is 'methyl and R is acetyl) is the most abundant. The major component of PIA-aura homomalla (Esper), 1792, forma S is PGA acetate methyl ester, the Formula-1X compound wherein R is methyl and R is acetyl. Relatively minor components are the hydroxy methyl ester. the acetate, and the hydroxy acid of Formula IX, and the PGE compounds corresponding to Formula Vlla but having the (5) configuration. v v k I When the acetate methyl ester compound of Formula VIla, V111, or 1X R is methyl, 12 1s acetyl) is desired as a starting material, a suitable method comprises grinding the frozen while Plexaura homomalla colonies or colony pieces, advantageously in a hogger toa particle size with the largest dimension about 5 mm., and then extracting the resulting particles with any of the usual organic solvents, preferably one with moderate to high polarity, elg., dichloromethane or methanol, advantageously, for 15 to 30 minutes in a high speed mixer. The desired compounds are isolated from the extract by evaporation, and then chromatography of the resulting residue. By this procedure, about 24 g. of lSB-PGA acetate methyl ester, and about 1 g. each of 15,8-PGA methyl ester and 15,8-PGE are obtained by dichloromethane extraction of 1500 g. of frozen Plexaura homomalla (Esper), l792, forma R Colonies Or colony pieces. Similarly, relatively large amounts of PGA acetate methyl ester are obtained from frozen Plexaura lwniomalla (Esper), l792, forma S.

When the lS-hydroxy methyl'ester of Formula Vlla, Vlll, or 1X (R is methyl, R is hydrogen) is desired as a starting material, a suitable method comprises grinding the frozen whole Plexaura homomalla colonies or colony pieces as above,and then contacting the resulting particles with a lower alkanol, preferably methanol or ethanol, at 25 C. for several days. The solvent is then evaporated and'the residue chromatographed to give substantially larger amounts of the hydroxy methyl ester compound relative to the acetate methyl ester compound. When the contact between the Plexau'ru lmmmnulla particles and the alkanol is substantially shorter, substantially the same amount and ratio of the various Formula-Vlla, -Vlll, or -IX compounds is obtained with the alkanol as with dichloromethane. An alternative method for' obtaining these l5-hydroxy methyl esters is described below.

When 15B-PGA2,'15,8-P,GE2, or PGAZ R; and R, in

Formulas Vlla, V111, and 1X are both hydrogen) are de sired as starting materials in the novel processes of this invention, they are prepared from the corresponding methyl esters and lS-acetate methyl esters after those have been extracted from the Plaraura homomalla colonies or colony pieces as described above. A suitable method for removing the acetyl group of each of the Formula-Vlla, -Vlll, and -IX l5-acetate methyl esters comprises mixing the acetate methyl ester in lower alkanol solution, preferably in methanol solution, with a strong acid, e.g., perchloric acid, for about 15 hours at 25 C. A suitable method for removing the methyl group of any of the Formula-Vlla, -Vlll, and -IX methyl esters is the enzymatic hydrolysis described in West Germany Qffeniegungschrift No. 937,912, reprinted in Farmdoc Complete Specifications, Book No. 14, No. 686911, Week R Mar. 18, 1970.

Another method for obtaining l5B-PGA ISB-PGE- or PGA from Plexau ra homomalla colonies or colony pieces comprises freezing the Plexaura homomalla colonies or colony pieces, preferably at a temperature below about 20 C., and then allowing the colonies or colony pieces to thaw and warm to a temperature in the range 20 to 30? C. The thawed colonies or colony pieces are then maintained in the range 20 to 30 C. for at least 24 hours. After that treatment, substantially none of the Formula-Vlla, -Vlll, and -IX compounds wherein R is methyl and R,, is acetyl are present, the principal Formula-Vlla, -Vlll, and -lX I compounds present being those wherein R and R are both hydrogen, the minor components being those wherein R is methyl and R is hydrogen or wherein R is hydrogen and, R is acetyl. As before, Formula-Vlla and -Vlll compounds are obtained from colonies of Plexaura homomalla (Esper), 1972, forma R, and Formula-IX compounds are obtained from colonies of Plexaura homomalla Esper), 1792, forma s.

A preferred procedure for the PGA and PGE type free acids comprises grinding the Plexaum 'llumumalla colonies or colony pieces, preferably to a particle size with the largest dimension about mm., and'then maintaining the mixture in contact with water at a temperature in the range to C. for at least 24 hours. This mixture is filtered, and the filtrate is extracted with an appropriate water-immiscible solvent, e.g., ethyl acetate. The solid residue is also extracted with an appro priate solvent, e.g., methanol. The two extracts are evaporated, and the total residue is chromatographed -to give Formula-VIIa and -VIII or Formula-IX compounds, the principal component in each case being the compound wherein R and R are both hydrogen.

Since our invention of the novel processes for transforming PGA and l5B-PGA and their methyl esters and acetate methyl esters to the various prostanoic acids and esters disclosed herein, it has now been found that small amounts of the 5,6-trans compounds of PGA and l5/3-PGA and their methyl esters and acetate methyl esters are also obtained from Plexaura lmmumalla (Esper), 1792, forms R and S. These 5,6- Trans compounds are extracted with and accompany the corresponding PGA- -type compounds through many of their transformations. For example, FGA containing 5,6-trans-PGA yields a mixture of PCE and 5,6-transPGE by the process represented in Chart E below.

When it is desired, for pharmacological purposes, to prepare the major products of this invention free of 5,6-trans compounds, those 5,6-trans compounds are separated either from the starting reactants or from the products. In either case, several methods are available for separating the 5,6-trans-PG compounds from the PG compounds. One method is by means of a silversaturated ion-exchange resin (for example, see E. A. Emken et al., J. Am. Oil Chemists Soc. 41, 388 1964)), illustrated below in Preparations 5 and 6. The other method is by preferentially forming a mercuric acetate adduct of the 5,6-cis compound which is extractable into polar solvents illustrated below in Prepa ration 7.

Following the processes discussed herein and the procedures of the Examples below, the 5,6-trans-PG (and -l5B-PG compounds are transformed to other 5,6trans-PG (and -l5,B-PG compounds, e.g., 5,6- trans-PGA to 5,6-trans-PGE 5 6-trans-15B-PGA acetate methyl ester to 5,6-trans-l5B-PGF a acetate methyl ester, and the like.

As mentioned above, the Formula-Vlla, -VIII, and -IX compounds are starting materials for the preparation of PGE PGFza, and PGF the methyl esters of those, and also the novel compounds of Formulas V and VI, and some of the novel compounds of Formula VII. The novel processes using these starting materials will now be described.

The Formula-VIII and -IX starting materials are both of the PGA-type. According to the novel processes of this invention, those are first transformed to corresponding PGE-type compounds. The chemical reactions involved in those transformations are shown ge-.

nerically in Chart A.

In Chart A, R is hydrogen, methyl, or -Si-(A) wherein A is alkyl of one to 4 carbon atoms, inclusive, aralkyl of 7 to 12 carbon atoms, inclusive, phenyl, or phenyl substituted with one or 2 fluoro, chloro, or alkyl of one to 4 carbon atoms, inclusive; G is wherein R is hydrogen, acetyl, or -SI-(A) when R, is hydrogen or methyl, and R is -Si(A) when R, is -Si- (A);,; R: is hydrogen or methyl; and B is t I or H OH.

wherein R, is as'de'fined above, and Z is wherein A is as defined above.

In Formula XI, of Chart A, indicates attachment ofthe epoxy oxygen tothe ring in alpha or beta configuration. In Formulas XI] and Xl'll ofiChart A, indicates attachment of hydroxy to the ring in alpha or beta configuration.

CHART Ac (oxidation) (reduction) Xll CHART A-Continued (hydrolysis) Xlll i.e., l lB-PGE and l IBJSB-PGE if the Formula-Xll or -Xlll productis to have the l5(S) configuration, e.g., PGE or I IB-PGE then the Formula-X starting material should have the l5(S) configuration, i.e., G

should be I If a I513 compound of Formula Xll or XIII is desired, e.g., 15,8-PGE or 1 l,8,l5,8-PGE then the Formula-X starting material should have the (R) or l5-epi configuration. i.e., G should be As described above, Formula-IX starting materials wherein R is hydrogen or methyl and R is hydrogen. and with the l5(S) configuration, are obtained from Plexaura lmmmnullu (Esper), l792, forma S. Those same compounds are also produced by reacting the corresponding 15(R) (beta) compound with a hydrocarbyl or halohydrocarbyl sulfonyl chloride or bromide, preferably a lower alkylsulfonyl chloride or bromide, especially methanesulfonyl chloride or bromide, or a benzeneor substituted-benzenesulfonyl chloride or bromide, e.g., p-toluenesulfonyl chloride. This reaction is done in the presence of at least sufficient tertiary amine, e.g., triethylamine, to absorb the hydrogen chloride or hydrogen bromide by-product, and at a low temperature, preferably in the range 15 to +15 C. The presence of an inert liqiud diluent, e.g., tetrahydrofuran, is helpful to maintain a mobile homogenous re action mixture. At 0 C. and with methanesulfonyl chloride, usually to 60 minutes is a sufficient reaction time. The product is hydrolyzed to a mixture of 15(8) (alpha) and ,l5(R) hydroxy compounds. These are separated by procedures known in the art, and the l5(S) productis purifiedby procedures known in the art, advantageously by chromatography on silica gel. This reaction is also used to transform l5(S) Formula- IX starting materials wherein R is hydrogen or methyl and R is hydrogen tothe corresponding 15(R), compounds. In each case, a mixture of l5(R) product and l5(S) starting material is obtained, the components of which are separated as described above.

Another method of transforming a lSfi-PG compound to a PG compound is by converting it to a mixture of PG l5-formate and ISB-PG l5-formate compounds, separating the PG l5-formate, and hydrolyzing the PG l5-formate to the desired PG compound (see JpE. Pike et al., J. Org. Chem. 34, 3552 (1969)).

The mixture of alpha and beta l5-formates is prepared by maintaining the 15/3 compound, e.g. 15B- PGE 15B-PGA or l5B-PGF a in formic acid buffered with an alkali metal formate in the range 10 to C. until a substantial amount of the 158 compound, e.g. ISB-PGE l5-forrnate, has been transformed to the PG l5-forrnate. The mixture of the PG l5-formate and 15B 15 -formates thus obtained is then separated by known methods, e.g. by chromatography. 9

The PG l5-formate can then by hydrolyzed to the desired PG lS-hydroxy compound. The 15,8-PG l5- formate yields the corresponding [SB-PG l5-hydroxy compound, which is then recycled through the above steps for further isomerization to the PG compound if desired. 7

This procedure is also useful to transform a PG compound to a 153 PG compound, by obtaining a mixture of the intermediate PG lS-formate and lSB-PG l5- formate compounds, separating them, and hydrolyzing them to the respective PG l5hydroxy and lSB-PG l5- hydroxy compounds. In this case, the PG compound is recycled for further isomerization to the 158 compound.

Referring again to Chart A, the transformation of starting material X to epoxide X! is carried out by reacting X with any agent known to epoxidize an aB-unsaturated ketone without reacting with isolated carboncarbon double bonds, for example see Steroid Reactions, Carl Djerassi, ed., Holden-Day Inc., 1963, p. 593. Especially preferred are aqueous hydrogen peroxide or an organic tertiary hydroperoxide. See, for example, Organic Peroxides, A. V. Tobolsky et al., lnterscience Publishers, N.Y., 1954. For this purpose, the peroxide or hydroperoxide is employed in an amount of at least one equivalent per mole of Formula-X reactant in the presence of a strong base, e.g., an alkali metal hydroxide, a metal alkoxide, or a quaternary ammonium hydroxide. For example, there is employed lithium hydroxide, sodium hydroxide, potassium hydroxide, lith ium ethoxide, ithium octyloxide, magnesium methoxide, magnesium isopropoxide, benzyltrimethylammonium hydroxide, tetraethylammonium hydroxide, butylrimethylammonium hydroxide, butyldiethylphenylammonium hydroxide, benzylethyldimethylammonium hydroxide, benzyldimethyloctadecylammonium hydroxide, benzyldodecyldimethylammonium hydroxide, decyldimethylphenylammonium hydroxide, and the like. See, for example, Sidgwick. Organic Chemistry of Nitrogen, Third Edition, rev. by Miller and Springall, Oxford, 1966, pp. 1 l6l27.

The ratio of alpha to beta epoxide formed in the reaction is related to four factors: the epoxidizing agent, the base, the diluent, and the temperature. Hydrogen per; oxide is employed in the concentrations usually available, for example 3% to 90%, although 30% is espel,1-diphenylethyl hydroperoxide. The base is present in the proportion of 0.1-3.0, preferably about 0.1-0.5 equivalent of base per mole of starting material X when R, is methyl and R is acetyl; preferably about 1.5-2.5 equivalent of base per mole of starting material Vlll or IX wherein R and R are hydrogen. When the alpha epoxide is the desired product, lithium hydroxide, lithium or magnesium alkoxides of one to 8 carbon atoms, and benzyltrimethylammonium hydroxide are the preferred bases, although the lithium and magnesium compounds are especially preferred.

It is advantageous to use an inert liquid diluent in the epoxidation step to produce a mobile homogenous reaction mixture, for example, a lower alkanol, dioxane, tetrahydrofuran, dimethoxyethane, dimethylsulfoxide, or dimethylsulfone. When the alpha epoxide is preferred, tetrahydrofuran or the less polar dimethoxyethane are especially preferred as the diluent. A reaction temperature in the range 60 to C. is generally preferred, especially below l0 C. The lower temperatures below 30 C. are especially preferred for favoring formation of alpha epoxide over beta epoxide. At a temperature of 20 C., the epoxidation is usually complete in 3 to 6 hours. It is also preferred that the reaction be carried out in an atmosphere of an inert gas, e.g., nitrogen, helium, or argon. When the reaction is complete as shown by the absence of starting material on TLC plates (3% acetone in dichloromethane), the reaction mixture is neutralized, and the epoxy product is isolatedby procedures known in the art, for example, evaporation of the diluent and extraction of the residue with an appropriate water-immiscible solvent, e.g., ethyl acetate. I

This transformation of to X] usually produces a mixture of Formula-XI alpha and beta epoxides both with either the 15(R) or l( S) configuration depending on the configuration at C-l5 in the Formula-X starting material. Although these mixtures are separated into the individual alpha and beta isomers, for example, by chromatography by procedures known to be useful for separating alpha and beta epoxide mixtures, it is usually advantageous to transform the Formula-XI mixture of alpha and beta epoxides to the corresponding mixture of Formula -XI[ 1 la and 1 1,8 hydroxy compounds. The latter mixture is then readily separated into the i la and l 1,8 compounds, for example, by chromatography of silica gel.

During the transformation of epoxides Xl to alcohols Xll, an alpha epoxide yields an i lac-hydroxy compound, and a beta epoxide yields an 1 lB-hydroxy compound. The ratio of alpha to beta epoxides in X], and hence the eventual ratio of Ila and 11B alcohols in XII, produced from starting material X is dependent in large measure on the nature of R in X. Recall that G is defined as wherein R is hydrogen, acetyl, or 'Si"( ):l wherein A is as defined above. For either definition of G, i.e., R configuration of S configuration, when R is hydrogen, more Formula-XI beta epoxide: is formed than when R is acetyl, and more Formula-XII beta epoxide is formed when R, is acetyl than when R, is -Si-(A),,. For example, when G in formula X is the preferred basic hydrogen peroxide epoxidation gives about equal amounts of alpha and beta epoxides,

but when G in formula X is I H OCOCH about 3 parts of alpha epoxide and one part of beta epoxide are obtained, and when G in formula X is .343

about 4 parts of alpha epoxide and one part of beta epoxide are obtained, both reactions with the same epoxidation reagent. When G in formula X is about one part of alpha epoxide and 3 parts of beta epoxide are obtained with basic [hydrogen peroxide, but. i y

when G is about 6 parts of alpha epoxide and 4 parts of beta epoxide are obtained with the same epoxidation reagent.

Each of the novel Formula-V and VI compoundsof this invention has a hydroxy attached in beta configuratiaon to the cyclopentane ring. Some of the compounds of Formula V, i.e., when Y is On the other hand, when a. prostanoic acid product with the natural 1 lo: configuration for the hydroxy at C-ll is thedesired final product,.e.g.,- PGE PGFQa, or PGFgB, there is advantage in choosing a Formula- X starting material which gives a greater amount of the alpha epoxide during the transformation of X to XI. Those would be Formula-X'compounds wherein X is or the'corresponding 15(S) compounds I H I As mentioned above, the startingmaterials of Formula X encompass not only the Formula-VIII and -IX compounds obtained from Pla 'uuru Immomulla but also the silyl compounds of Formula Xa. When desired as reactants, these silyl compounds are prepared by silylation of PGA ISB-PGA- or the methyl esters of those. These silylations are carried out by procedures knonn in the art. See, for example, Pierce Silylatio'n of Organic Compounds, Pierce Chemical Co., Rockford, Ill. (1968). The C-l hydroxy group of PGA;;,] 5B-PGA- or their methyl esters is transformed to an -O--Si(A) moiety wherein A is as defined above, sufficient silylating agent being used according to known procedures to accomplish that. The necessary silylatingagents for this purpose are known in the art or are prepared by methods known in the art. See, for example, Post, Silicones and Other Organic Silicon Compounds," Reinhold Publishing Corp., New York, N.Y. 1949 In the case r PGA and 15;;- PGA. excess silylating agent and prolonged treatment also transform the -COOH to COOSi(A);,. It is optional in transforming X to XI whether or not this ,'COOH- of PGA or l5B-PGA *is esterified to COOSi(A);,, since that ester group is transformed to COOH during formation and isolation of the Formula-XI epoxide product. 7

,The various As of a -Si-'(A).-; are alike or different. For example, an Si(A);; can be trimethylsilyl,

dimethylphenylsilyl, or methylphenylbenzylsilyl.

When it is desired to retain the ,Si (.A) moiety at C-l5 in the Formula-XI epoxide product, for example, to give steric control in a subsequent reaction, it is important in isolatingthe epoxide that ,the presenceof acid be avoided and that contact with water be minimized unless the water is kept cold, i.e., below about 10 C.

Referring again to Chart A, the transformation of epoxide XI to hydroxy compound XII is accomplished by reduction with chromium (II) salts, e.g., chromium (II) chlorideor chromium (II) acetate. Those salts are prepared by methods known in the art, e.g., Inorganic Syntheses, VIII, 125 (1966); ibid., VI, 144 (1960); ibid. 111, 148 (I950); ibid. l, 122(1939); and references cited in those. This reduction is carried 'out by procedures known in the art for using chromium (II) salts to reduce epoxides of ozB-unsaturated ketones 'to ,B-hydroxy ketones. See, for example, Cole et al., J. Org. Chem, l9, 131 (I954), andNeher et all, Helv. Chem. Acta 42, 132( 1959). In these reactions. the absence-of air and strong acids is desirable. If it is desired to maintain a Si-(A),, moiety on C-l5, a neutral reaction mixture is preferred. An especially preferred procedure is to generate the chromium (II) ion in the presence of the Formula-XI epoxide, for example, by mixing the epoxide with a chromium (III) salt, e.g., the chloride, with metallic zinc in the presence of acetic acid. The desired Formula-XII compound is isolated from the reduction reaction mixture by methods known in the art, care being taken to minimize contact of the product with acid and water, especially warm water, when retention of a Si (A) at C-l5 is desired.

Unexpectedly, amalgamated aluminum metal has also-been found to be useful as a reducing agent in placeof chromium (II) salts to transform Formula XI epoxidesto Formula XII hydroxy compounds. This reagent was previously not known to be useful for this type of reaction. This use of amalgamated aluminum represents a distinct and separate aspect of this invention.

Amalgamated aluminum is prepared by procedures known in the art, for example, by contacting aluminum metal in the form of foil, thin sheet, turnings, or granules with a mercury (II) salt, for example, mercuric chloride, advantageously in the presence of sufficient water to dissolve the mercury (II) salt. Preferably, the surface of the aluminum metal is free of oxide. That is readily accomplished by physical removal of the usual oxide layer, e.g., byabrasion or scraping, or chemically, e.g., by etching with aqueous sodium hydroxide solution. It is only necessary that the aluminum surface be amalgamated. The amalgamated aluminum should be freshly prepared, and maintained in the absence of air and moisture until used.

The reduction opening of the Formula-XI epoxide ring is accomplished by'contacting said epoxide .with the amalgamated aluminum in the presence of a hydroxylic solvent and sufficientinert organic liquid diluent togive a mobile and homogeneous reaction mixture with respect to the hydroxylic solvent and said epoxide. Among hydroxylic. solvents, water isv especially preferred although lower alkanols, e .g., methanol and ethanols are also operable.

Examples of inert organic liquid diluents are normally liquid ethers such as diethyl ether, tetrahydrofuran, dimethoxyethane, diglyme (dimethylether of diethylene glycol), and the like. Especially preferred is tetrahydrofuran. When a water-immiscible liquid diluent is used, a mixture of water and methanol or ethanol is especially useful in this reaction since the latter two the hydroxylic solvent to give 3 atomic equivalents of hydrogen. Therefore, one molecular equivalent of epoxide requires two-thirds atomic equivalent of aluminum and two molecular equivalents of the hydroxylic solvent. Evolution of hydrogen gas (I-I molecules) is observed during this reductive opening of the epoxide. It is not'known whether the reductive opening is caused by hydrogen atoms or hydrogen molecules. However, some of the hydrogen gas escapes from the reaction mixture. Therefore, it is preferred to use an excess of amalgamated aluminum and hydroxylic solvent, preferably at least one atomic equivalent of aluminum and three molecular equivalents of hydroxylie solvent per molecular equivalent of epoxide. Because of the relatively high economic value of the epoxide compared with amalgamated aluminum and hydroxylic solvents, it is preferred to assure maximum yields of the desired Formula-XII hydroxy compound by use of substantially greater excess of amalgamated aluminum and hydroxylic solvent, e.g., up to ten times or more of those reagents than is theoretically'required.

The reductive opening of the epoxide is carried out by mixing a solution of the epoxide in the organic diluent with the amalgamated aluminum and the hydroxylic solvent. Since the reaction is exothermic, it is usually advantageous to cool the solution to a low temperature, e.g., 20 C. to C., before adding the amalgamated aluminum and hydroxylic solvent and to maintain the reaction mixture in the reaction mixture in the range to C. by external cooling. This is especially advantageous when water is used as the hydroxylic solvent. Higher reaction temperatures are operable but not preferredwhen a high yield of the Formula-XII products is desired. Stirring is preferred during' the reaction since the reaction mixture is heterogeneous with respect to the solution and the amalgamated aluminum.

For reasons not understood, better yields and a shorter reaction time are usually observed when only part of the amalgamated aluminum is added at the start of the reaction, additional portions being added during the re'action, e.g., at one-hour intervals, than when the entire amount of amalgamated aluminum is added at the start of the reaction. A suitable procedure is to add about one-third of the amalgamated aluminum at the start, about one-third after one hour, and another third after a second hour. The course of the reaction is advantageously determined by withdrawing small portions of the solutionand determining thepresence of absence of starting material by thin layer chromatography. For example, when R: is methyl and G is Xla ii, f

are as defined above and R is a cation of an alkali or alkaline earth metal or a quaternary ammonium group.

Thusjthe Formula-XI epoxide compound is treated with a hydroxide or oxide of lithium sodium, potassium, magnesium, calcium, barium, or strontium prior to contacting with the aluminum amalgam. Optionally, the quaternary ammonium bases are used for this neutralization, for example benzyltrimethylammonium hydroxide. The base is used in equivalent amount to the acid so that R is replaced by the corresponding metal or quatemary-ammonium cation. Alternatively, instead of the hydroxides or oxides, there are employed the hydrides, the carbonate, the bicarbonates, or the alkoxides, for example lithium hydride, potassium carbonate, sodium bicarbonate, magnesium methoxide, and the like, which form the corresponding Formula-Xla slats with the Formula-XI free acid. Alternatively, a metal or quaternary-ammonium carboxylate compound or Formula-Xla salt carriedforward from the e'poxidation step, whether isolated in that step or not, is employed in the reductive step with aluminum amalgam. It is preferred that the'Formula-Xla salt be soluble in the organic diluent-alkanol-water or organic diluentwatermedium used for the reduction step. By using the above-described salts, the reduction step proceeds smoothly without formation of insoluble aluminum salts which hinder the reaction. Following the reduction or hydrolysis step, the R cations are replaced with hydrogen by means known in the art, for example by acidification and extraction of the acid compound into an organic phase.

The desired Formula-XII hydroxy products are isolated by filtration of the reaction mixture. advantageously after addition of magnesium sulfate as a filter aid, and evaporation of the organic diluents. The Formula-Xll products are then hydrolyzed if desiredto' remove Si (A);i from C-l5, and the 11a and 113 products of Formula'Xlll are separated, if desired, by procedures known in the art, e.g., chromatography on silicagel.

The products of Formula-X1] are all of the PGE -type and include PGE PGE lS-aeetate, PGE methyl ester, PGE l5-acetate methyl ester, PGE and PGE methyl ester with an -OSi-(A).-; at C-l5, the corresponding 15B compounds. and compounds corresponding to all of those whereinhydroxy is attached to C11 in beta configuration.

As mentioned above, the transformation of X to X] to Xll usually gives a mixture of Formula-XII PGE-type products, part with alpha and part with beta configuration for the hydroxy at C-1 1. There are several alternatives regarding that mixture. If -O-Si- (A)3 is attached to C-l5, that can readily be transformed by hydrolysis to -OH. These hydrolyses are carried out by prior art procedures known to be useful for transform ing'silyl ethers to alcohols. See, for example, Pierce, cited above, especially p. 447 thereof. A mixture of water and sufficient of a water-miscible organic diluent to give a homogeneous hydrolysis reaction mixture represents a suitable reaction medium. Addition of a catalytic amount of an organic or inorganic acid hastens the hydrolysis. The length of time required for the hydrolysis is determined in part by the hydrolysis temperature.

wherein G and With a mixture of water and methanol at 25C., several hours is usually sufficient for hydrolysis. At C.. several days is usually necessary. Also, i --OCOCH;, is attached to C-l5. that can readily be transformed to ---OH by acid-catalyzed alcoholysis as described above for removing the acetyl group of the Formula-VIII and -IX PGA-type starting materials. Both of those transformations are shown in Chart A, i.e.. XII to XIII. Either before or after those transformations of XII to XIII, the Formula-XII or -XIII mixture of l la and 1 1p! isomers can be separated by methods known in the art, advantageously by chromatography on silica gel.

Further regarding the Formula-XIII compounds, those compounds wherein the configuration of the hydroxy at C-l l is beta are within the scope of the Formula-V novel compounds of this invention, and those compounds wherein the configuration of the hydroxy at C-1 l is alpha and B is are within the scope of the Formula-VII novel compounds of this invention. Both groups of novel compounds are used for pharmacological purposes described above forthose compounds, the acids also being useful as reactants to prepare pharmacologically useful esters and pharmacologically acceptable and useful salts, both as described above. Moreover, Formula-XIII compounds wherein the configuration of the hydroxy at C-l l is alpha and B is are F'GE and PGE methyl ester, both of known pharmacological utility.

Still further regarding the separated compounds of Formulas XII and XIII, when a compound with one configuration at C-l 1, either alpha or beta, is desired as an intermediate or for pharmacological purposes, the other isomer is readily dehydrated to give additional Formula-X PGAtype starting material which is then used as a starting material according to the processes defined in Chart A and procedures described above to give additional of the desired isomer. These dehydrations are accomplished by procedures known in the art for dehydration of PGE-type compounds to PGA-type compounds. See, for example, like et al., Proc. Nobel Symposium 1 I. Stockholm 1966), Interscience Publishers, New York, p. I62 1967), and British Specification 1,097,533. These are acidic dehydrations, and alkanoic acids of 2 to 6 carbon atoms, inclusive, especially acetic acid, are preferred for this purpose. Dilute aqueous solutions of mineral acid, e.g., hydrochloric acid, especially in the presence of a solubilizing diluent, e.g., tetrahydrofuran, are also useful as reagents for these acidic dehydrations, although these reagents may also cause partial hydrolysis of the Formula-XII or XIII methyl esters to carboxylic acids. ASi-(A) moiety at C-l5 is also removed during all of these acidic dehydrations.

Still further regarding the Formula-XII and -XIII. compounds, either as mixturesor separately, any of those is transformed to other useful compounds or mixtures by changing these PGE-type compounds to PGF- type products by reducing the ring carbonyl at- C-9 to alpha hydroxy or beta hydroxy. Those transformations are shown in Chart B.

In Chart B, R, is hydrogen. methyl, or Si(A)a. R is hydrogen or methyl, R is hydrogen or -Si(A):,,

and G is H 0R or 0R wherein R is hydrogenor SI (A):I when R and R are hydrogen; R is hydrogen, acetyl, or -Si(A) when R and R are methyl; and R is Si(A-).-; when R, is Si(A);;, wherein A is as defined above, with the proviso that when R, is .-Si(A) R is also -Si- --(A);;. Further in Chart B, B in Formula XVI is or H OI I,

H OH

CHART B cM/Ymog XH (si lylation) XIV (reduction) (hydrolysis or 'alcoh oiysis) CHART B Continued cooa,

Referring to Chart B, the starting material XII can be a mixture of compounds with regard to the configuration of G1 I, or the starting materialcan be stereochemically pure with respect to 01 l, depending upon whether there has been an earlier separation of I10: and 1 1B isomers (see above discussion of Chart A reactions).

The transformation of PGE-type starting material XII to PGF-type product XVI involves reduction of a ring carbonyl to a ring hydroxy. This process is known in the art for some of the compounds encompassed by Formula XII, i.e., when the configuration at C-ll is alpha and the configuration at C-l5 is S. For the other compounds encompassed by Formula XII, this reaction is novel, and novel Formula-XV and -XVI compounds are produced.

For this carbonyl-to-hydroxy reduction, methods known in the art are used. See, for example, Pike et al., J. Org. Chem 34, 3552 (1969). Use is made of any of the known ketonic carbonyl reducing agents which do not reduce ester or acid groups or carbon-carbor double bonds. Examples of those are the metal borohydrides, especially sodium, potassium, lithium, and zinc borohydrides, lithium (tri-tert-butoxy) aluminum hydride, metal trialkoxy borohydrides, e.g., sodium trimethoxyborohydride, and diisobutylaluminum hydride. The sodium, potassium, and zinc borohydrides are preferred for this reduction, especially zinc borohydride.

Unexpectedly, the amalgamated aluminum metal found useful above in transforming the Formula-XI epoxides to Formula-XII hydroxy compounds has also been found useful as an agent for this carbon yltohydroxy reduction of PGE-type compounds to PGF- type compounds. Either the PGE-type salts or the PGE- type esters are employed, for example the Formula-XII hydroxy compounds produced from the Formula-XI epoxides with or without intermediate isolation. Furthermore, the Formula-XI epoxides may be subjected to the combined epoxide-reduction and carbonylreduction reactions practically simultaneously by operating at higher temperatures, for example 40-60 C.,

although it is preferred for high yields of the 11- hydroxy compounds that the reductions be done stepwise. The solvents which are operable for this reduction are generally the same as those found useful for the epoxide-reduction step. Somewhat higher temperatures or longer reaction times are required for the carbonyl-to-hydroxy reduction, however. For example, at 25 C., about 4 to 24 hours are required; at higher temperatures, e.g., 5060 C., about one to 2 hours are sufficient.

This carbonyl reduction usually produces a mixture of PGF -type and PGF -type compounds, i.e., compounds with the alpha configuration and compounds with the beta configuration for the hydroxy at C-9. This mixture of alpha and beta isomers is separated by methods known in the art, e. g., chromatogra- XVI phy on silica gel. See Pike et al., ibid., for example. If the Formula-XII starting material is a mixture oi Ila and 1 1/3 isomers. then this reduction will usually produce four isomers, i.e., 9a,lla,9a,ll,8,9fi,l la. and QBJ l8. Those compounds are also separated from such mixtures by silica gel chromatography.

Regarding the transformation of Xll to XIV in Chart B, it will be observed that the parameters for XII are such that all XII compounds are included in XIV. In other words, the transformation XII to XIV is an optional process step in proceeding from XII-to XV. The reason for this is as follows. During the reduction of XIV to XV, the ratio of Ba-hydroxy and 9B-hydroxy compounds formed will be different when R, in XIV is hydrogen than when R, is -SI-(A):I* For example, with the Formula-XIV compound wherein R, is hydrogen, G is and R O- represents I-IO'" i.e., l lot-hydroxy, sodium borohydride reduction gives -42 parts of the corresponding Formula-XV 9a-hydroxy compound, and 58 parts of the QB-hydroxy compound. But with the Formula-XIV compound wherein R, is hydrogen, G is and R O- represents (CH;,);,SiO'" sodium borohydride reduction gives parts of the corresponding Formula-XV 9a-hydroxy compound and 15 parts of the QB-hydroxy compound. Similar differences are observed with the other isomers encompassed by formula XIV although not necessarily in the same direction. Accordingly, whether R in Formula XIV is to be hydrogen or Si(A),-, depends on the particular formula XV C-9 isomer desired and the influence of silylation on the isomer ratio. For any particular Formula-XIV starting material, the latter is readily determined by small scale reduction with and without silylation. When silylation before carbonyl reduction is indicated, largely for economic reasons, it is preferred that A be i.e., R1 be (CH3)3'SI-. i

This transformation of XII to XIV wherein R is -Si- -(A),, is carried out as described above for the transformation of hydroxy to O-Si ):I at C-lS prior to the Chart A reactions. When R in XII is hydrogen, the -COOH is also transformed in part or entirely to ,COOSI(A):I with prolonged silylation and excess silylating agent. It is optional in transforming XII to XIV wherein R is Si--(A);, whether or not the -COOH' of XII is esterified to COO-Si( A) When 6 in Formula XIV is those OH are also transformed to O-SI(A)3 by this silylation.

With regard to the Formula-XV carbonyl reduction product (Chart B), when the method used to isolate said product does not remove any Si(A);, groups which may be present, that is accomplished as described above for the removal of -SI '(A)3 groups H OCOCH or H OCOCH.

the acetyl is removed by alcoholysis also as described above for-changing acetoxy at C-l5 to'hydroxy. These reactions are shown in Chart B as XV to XVI.

-When R in Formula XVI is methyl and the compound wherein R is hydrogen is desired, that methyl ester is saponified, by methods known in the art. See. for example, Just et al., J. Am.,-Chem. Soc. 91, 5371 (1969). This saponification also changes, a C-l5. ace tate to a'C-l5 hydroxy.

The compounds encompassed by Formula XVI include the known compounds PGFza, P6P and the methyl esters of those. Also included in Formula XVI are the novel compounds l5a-PGI a 15,8- PGF and the methyl esters of those. All of these new and old compounds are l lot-hydroxy compounds. Also included in Formula XVI are the corresponding but novel llfi-hydroxy compounds which are also encompassed by Formula V and which are useful for the pharmacological purposes described above either as such or transformed into salts or'esters as described above.

When one of these formula-XVI compounds has the R or epi configuration for the hydroxy at C-l5, and the corresponding compound with the S configuration at C-l5 is desired, or when one of these Formula-XVI compounds has the S configuration for the hydroxy at C-IS, and the corresponding compound with the R or epi configuration at C- l 5 is desired, those desired compounds are made by the process of Chart C. In Chart C, R ,*R R;, B, and 5' are as defined above.

The overall process scheme of Chart C is to start with one particular C-l5 isomer of a compound encompassed by Formula XVI, i.e., either l5(S) or (R). The C- l 5 hydroxy 'of that isomer is oxidized to'a-ketonic carbonyl (XVII). Thenfafter an optional silylation of the C-9 and C-l'l hydroxy'groups (XVIII), the C-l 5 carbonyl is reduced back to a secondary hydroxy group. That reductionproduces two C-'l5 hydroxy isomers, one with S configuration and one with R or epi configuration. After removal of any silyl groups, the isomers XIX and XX are separated. One of the isomers will be the same compound used as starting material (XVI). The other isomer will be the desired product. The starting material isomer is recycled to produce more of the desired isomer. This reaction scheme has previously been used to transform PCuF a to ISB- (oxidation) CHART C-Continued DDR2 XVll I (silylation) C 4 XVlll 1/ (reduction) h drol sis) w I J l y Y cook XIX H H. OH

Referring now to Chart C, starting material XVI (from Chart B) is a single compound, amixture'of two compounds, one with alpha and one with beta configuration at C-9, or a mixture of four compounds, i.e., 911,11, 901,115, 95,110., and 9B,] 13.

For the oxidation of XVI to XVII, any oxidizing agent can be used which will oxidize an allylic alcohol to an afi-unsattirated ketone or aldehyde. Examples of those are 2,3-dichloro-5,6-dicyano l,4 benzoquinone, activated manganese dioxide, or nickel peroxide (see F,ieser et 21],, fReagents for Organic Syntheses, John Wiley & Sons, Inc., New York, N.Y.,"I967, pp. 215, 637, and 731). Alternatively, these oxidations are carried out by oxygenation in the presence of the IS-hydroxyprostaglandin dehydrogenase of swine lung (see Arkiv for Kemi 25, 293 (1966)). Thesereagents are used according to procedures known in the art. See, for example, 1.1 Biol. Chem. 239, 4097 (I964).

Regarding the transformation of XVII to XVIII in Chart C, it will be'observed that the parameters for XVIII are such that all XVII compounds are included in XVIII. In other words, the transformation of XVII to XVIII is an joptional .process step in proceeding from XVII to XIX and XX. The reason for this is as follows. During the reduction of XVIII'to XIX and XX, the ratio of XIX to XX'obtained will be different when R in XVIII is hydrogen than when R is Si(A);,. For example, reduction of the Formula-XVIII 9a,1 lot-isomer wherein R and R are both hydrogen with zinc'borohydride gives the corresponding Formula-XIX and -XX in the amounts 43 parts of XIX (R or epi configuration) and 57 parts of XX (S configuration). On the other hand, when R; in the Formula-XVIII reactant is Si- (A);,, the amounts with the same reducing agent are 27 parts of XIX and 73 parts of XX. Similar differences are observed with the other isomers encompassed by Formula'XVIII although not necessarily in the samedirection. Accordingly, whether R in formula XVIII is to be hydrogen or Si-'-(A) depends on the particular C-l5 isomer desired and the influence of silylation on the isomer ratio. For any particular Formula-XVII starting material, the latter is readily determined by small scale reductions with and without silylation. When silylation before carbonyl reduction is indicated, largely for economic reasons, it is preferred that A be methyl, i.e., that R be (CH;,);,Si.

These silylations are carried out as described above for the Chart A and Chart B silylation.

The carbonyl reduction of XVIII to XIX iscarried out as described above for the transformation of PGE- type Formula-XIV compounds to PGF-type Formula- XV compounds. As for those reductions, the sodium, potassium and zinc borohydrides are preferred as reducing agents, especially zinc borohydride.

When the method used to isolate the carbonyl reduction product does not remove any Si-(A) groups which may be present, that is accomplished as described above for the removal of Si-(A) groups from Formula-XII products (Chart A, XII to XIII). The Formula-XlX and -XX products are separated from each other by methods known in the art, for example, silica gel chromatography. See, for example, Pike et al., J. Org. Chem. 34, 3552 1969) for this type of separation.

' If one of the isomers of isomer mixtures of Formulas XIX or XX is not desired for a pharmacological use as such or transformed to esters or pharmacologically acceptable salts as described above, that isomer or isomer mixture is recycled as a Formula-XVI starting material in the processes of Chart C to produce additional of the desired isomer. 7

The products of Formulas XIX and XX wherein the configuration of the C-1 1 hydroxy is beta are encompassed by Formula V. The products of Formula XIX wherein the configuration of the C-1 1 hydroxy is alpha are encompassed by Formula VI. The intermediates of Formula XVII are encompassed by Formula VII. Thus, all of the compounds are useful for the pharmacological purposes described above for the Formula V, VI and VII compounds. The compounds prepared as in Chart C are also useful to make the other esters and the pharmacologically acceptable salts of the Formula V, VI, and VII compounds also as described above.

There are two particular embodiments of the novel process of this invention which are especially preferred. One of those embodiments provides an optional route to PGF a and starts with 15,8-PGA acetate methyl ester, the most abundant component of Flexaura lmnwmaila (Esper), I792, forma R. The other embodiment provides a preferred route of PGE and starts with PGA readily obtained as described above by maintaining colonies or colony pieces of Plat-aura lwmoma/la (Esper), 1792, forma S in contact with water in a temperature range up to 50 C. until substantially free of PGA l5 acetate methyl ester.

The first of these embodiments is shown in Chart D, and the second is shown in Chart E. All of these Chart D and Chart E reactions and reagents for effecting them are described generically and specifically above, and all are exemplified below. In Charts D and E, it is preferred that Si-(A);, be Si (CI-I Also in Charts D and E, it is optional whether silylation of 15- oxo-PGF on (Chart D) or PGA (Chart E) produces the corresponding -'SI(A) ester-ether or only the ether.

CHART D l5B-PGA acetate methyl ester (oxidation) I5B-PGA acetate methyl ester (1 and [3 10,1 l-cpoxidcs v (reduction) ISIS-POE and l l/3,l5B-PGE lS-acctatc methyl esters 1, (separation) l5BPCiE l5-acctatc methyl ester 1 (silylation) ISBJGE l LSi-(A); ether IS-acctatc methyl ester 1 (reduction, hydrolysis) lSB-PGF a and lSfi-PGF IS-acctatc methyl esters l (separation) l5,8PGF J1 l5-acctatc methyl ester (saponification) 'l5/3-PGF a l (oxidation) lS-oxmPGlF (I (silylation) l5 oxo-PGF- a 9,] l-di-Si-(A);| (:thcr

(reduction hydrolysis) ISB-PGF a and PGF a (separation) i PGF CHART E PGAQ ltsilylation) PGA IS-Si-(Al cthcr (oxidation) PGA: l5-Si(A);, other a and ,8 l0 l l-cpoxidcs reduction, hydrolysis) PGE: and l II5-PGE uscparatiun) Poe; I

The invention is more fully understood by the following' Examples and Preparations All temperatures are in degrees centigrade.

Ultraviolet spectra are recorded on a Cary Model 15 spectrophotometer.

The collection of chromatographic eluate fractions starts when the eluant front reaches the bottom of the column.

Brine, herein, refers to an aqueous saturated sodium chloride solution.

The A-IX solvent system used in thin layer chromatography (TLC) is made up from ehtyl acetate-acetic acid-2,2,4-trimethylpentane-water (:20:50: l 00) according to M. Hamberg and B. Samuelsson, J. Biol. Chem. 241, 257 (I966).

Preparation 1: To distinguish Plexum-u lmmumallu (Esper), 1792, forma R from Plexuuru lzonmmullu (Esper), 1792, forma S, a TLC method is used. A specimen approximately 2 cm. in length is harvested and placed in a small vial, with a small amount of water if necessary to insure it is wet, and kept closed for 624 hrs. About 1 ml. of methanol is then added and the sample is either shaken for 2 hrs. at about 25 C. or is stored for 16-24 hrs. at about 10 C. A sample of the liquid (IO-21 A) is spotted on; a TLC plate. It is pre ferred to use a fluorescent-treated silica gel plate, e.g. Uniplate silica Gel GF (Analtech, Inc., Newark, Del.). As reference standards, spots of PGA and ISB-PGA are also applied. The plate is developed in the A-lX system (Hamberg and Samuelsson. J. Biol. Chem. 241. 257 1965)). The spots are finally visualized with vanillin-phosphoric acid spray (McAleer, Arch. Biochem. E. Biophys. 66, 120 (1957)). Comparison of the unknown with the two reference spots is then made and the identity of the coral established (forma S corresponding to PGA forma R to lfl-PGA Preparation 2: PGA from Plexaura homomalla (Esper), 1792 forma S.

Colonies of Plexaura homomalla (Esper), 1792, forma S, collected from reefs off the north shore of .lamica, are frozen by contact with solid carbon dioxide within one hour after removal from the reef waters. The frozen colonies are maintained in insulated boxes containing solid carbon dioxide (temperature below about 20 C.) until ready for thawing. Then, the frozen colonies (700 g.) are ground to a small particle size (Waring blender) and mixed with 1500 ml. of water. The mixture is maintained about 20 hrs. at about 25 C. with stirring. Then, the mixture is filtered through a pad of diatomaceous earth, and the filtrate is acidified with concentrated hydrochloric acid to pH about 2-3. The acidified filtrate is extracted four times with ethyl acetate. The extracts are'combined, filtered. washed with brine, dried with anhydrous sodium sulfate, and evaporated under reduced pressure to give 1 l g. of oily residue. I I

The solid residue on the diatomaceous earth filter pad is stirred 2 hours in methanol (enough to cover said residue) at 25 C. The mixture is then filtered, and the filtrate is evaporated to give 14 g. of oily residue.

The two oily residues are combined and chromato graphed on 1500 g. of acid-washed silica gel. eluting successively with 8 l. of a 25 to 657: gradient of ethyl acetate in Skellysolve B, 8 l. of a 65 to 100 gradient of ethyl acetate in Skellysolve B, and 5 l. of 2% methanol in ethyl acetate, collecting 500 ml. fractions. (Skellysolve B is a mixture of isomeric hexanes). Fractions 8-12 are combined and evaporated to give a small amount of PGA-; containing atrace of PGA methyl ester. Fractions -18 are combined and evaporated to give 9.54 g. of PGA Fractions 35-40 are combined to give 0.414 g. of PGE- Preparation 3: ISB-PGA from Plexaura Hmnomullu (Esper), 1792. forma R.

Colonies of Plexaura lzomomallu (Esper), 1792, forma R, collected from reefs off the southeast shore of Florida near Miami, are chopped into chunks several inches long. The chunks are frozen by contact with solid carbon dioxide with one hour after removal from the reef waters. The frozen colony pieces are maintained in insulated boxes containing solid carbon dioxide (temperature below about C.) until ready for thawing. Then, colony pieces (600 g.) are mixed with 1500 ml. of water. The mixture is stirred and maintained at C. for 23 hours. The mixture is then filtered through a pad of diatomaceous earth. and the filtrate is acidified to pH about 2-3 with concentrated hydrochloric acid. The acidified filtrate is extracted four times with ethyl acetate. The extracts are combined, filtered, washed with brine, dried with anhydrous sodium sultate, and evaporated to give 9.2 g. of oily residue.

The solid residue on the diatomaceous earth pad is stirred 15 hours in methanol (enough to cover said residue) at 25 C. The mixture is then filtered, and the filportions of 25%, 35%, 45%, 55%, and 65% ethyl acetate in Skellysolve B, collecting SOO-ml. fractions. Fractions 18-22 are combined and evaporated to give 5.54 g. of l5B-PGA Fractions 15-17 are combined and evaporated to give 1.37 g. of lSB-PGA methyl ester. Preparation 4: PGA compounds from Plexaura lwmonmlla (Esper), 1792, forma S.

Frozen colonies of Plexaura lzumomalla Esper), 1792, forma S (see Preparation 2) are broken manually into pieces several cm. in length. The pieces (500 g.) are then covered with methanol and the mixture is maintained for 3 hours at 25C. The mixture is then ground in a Waring blender and filtered, and the filtrate is evaporated under reduced pressure. The residue is dissolved in ethyl acetate, and the solution is washed successively with one N hydrochloric acid. water, and brine, dried with anhydrous sodium sulfate, and evaporated under reduced pressure. The oily residue is chromatographed on 2 kg. of acid-washed silica gel wetpacked with Skellysolve B (a mixture of isomeric hexanes), eluting with 24 l. of a 25 to 100% ethyl acetate in Skellysolve B gradient. The fractions which contain PGA acetate methyl ester, PGA: acetate, PGA methyl ester, and PGA. as shown by TLC with the A-IX system are separately combined and evaporated to give those compounds.

Preparation 5 lSfi-PGA compounds from Plexaura /I()I)IUHIU(I (Esper), 1792, forma R.

Colonies of Plexaura homomalla (Esper), 1792, forma R, collected from reefs off the southeast shore of Florida near Miami, are chopped into chunks several inches long. The chunks are frozen by contact with solid carbon dioxide within one hour after renoval from the reef waters. The frozen colony pieces are maintained in insulated boxes containing solid carbon dioxide (temperature below about 20 C.) until the time for extraction. Then, the frozen colony pieces are ground to a small particle size (Mitts and Merrill hogger; average largest dimension about 5 mm). The particles 1500 g.) are then stirred at high speed with 5 gallons of dichloromethane for 20 minutes at about 25 C. external temperature. The mixture of dichloromethane and particles is then filtered through a pad .of diatomaccous earth, and the filtrate is evaporated to about a 2- liter volume at 30 C. under reduced pressure. The liquid which remains is washed with water, dried with so dium sulfate, and evaporated at 30 C. under reduced pressure.

The oily residue (60 g.) is chromatographed on 3 kg. of silica gel wet packed in Skellysolve B (a mixture of isomeric hexanes), eluting successively with a gradient of4 l. of Skellysolve B and 4 l. of 20% ethyl acetate in Skellysolve B, 27 l. of 20%. 18 l. of 50%. and 8 l. of ethyl acetate in Skellysolve B, collecting 600-ml. fractions. Fractions 39-60 are combined and evaporated to give 243 g. of, l5B-PGA acetate methyl ester. Between fractions 60 and 74 those fractions shown by TLC to contain l5/3-PGA acetate are combined and evaporated to yieldthat compound. Fractions 74-76 are combined and evaporated to give 1.03 g. of 158- PGA methyl ester. Fractions 83-91 are combined and evaporated to give 1.08 g. of 15B-PGE lS-acetate methyl ester. Still later fractions shown by TLC to contain l5B-PGE methyl ester are combined and evaporated to yield that compound.

Detection of the respective compounds by TLC is done by methods known in the art, e.g. by Spotting the extract fractions on a TLC silica gel plate alongside spots of the authentic compounds, developing the plate with the A-IX system, and observing which spots of the extract fractions correspond exactly to the spots of the authentic compounds.

Following the procedures of Preparation 5, but substituting Plexaura homomalla (Esper), 1792, forma S for the Plexaura lwmomalla (Esper), 1792, forma R of that example, there are obtained the corresponding compounds of 15(S) configuration, viz.: PGA acetate methyl ester, PGA acetate, PGA methyl ester, PGE l5-acetate methyl ester, and PGE methyl ester.

Preparation 6: PGA and 5,6-trans-PGA Separation of PGA from 5,6-trans-PGA is done on a chromatographic column using a silver-saturated ionexchange resinl Preferably a macroreticular ion exchange resin is used, e.g. a sulfonated styrenedivinylbenzene copolymer having surface area of 40-50 sq. m./g., 30-40% porosity, and total exchange capacity of 4.5-5.0 meq. per gram of dry resin, for example Amberlyst 15, available from Rohm and Haas Co., Philadelphia, Pa. The acid-form resin is packed in a column, washed with warm water, and converted to the silver form by passing a 10% silver nitrate solution through the column until the effluent shows a pH of 3.5-4.0. The column is then washed with water to remove ionic silver, and finally with denatured ethanol (Type 3A). A solution of a mixture of PGA and 5,6- trans-PGA e.g. fractions -18 of Preparation 2, in ethanol is charged to the column. Elution with 3A alcohol then yields fractions which are combined according to their content of 5,6-trans-PGA (faster-eluting) or PGA Testing for the presence of 5,6-trans-PGA or PGA in the eluate is conveniently done by TLC using silver nitratetreated silica gel plates (e.g. Analtech Uniplates dipped in saturated ethanolic silver nitrate and dried) and developing with the A-lX system. R, of PGA is 0.45; R of 5,6-trans-PGA 2 is 0.50. Combined fractions are concentrated, partitioned between dichloromethane and a little water, dried over sodium sulfate, and concentrated under reduced pressure to yield the title compounds.

For quantitatively assaying the 5,6-trans-PGA content of mixtures of PGA and 5.6-trans-PGA a combination thinlayer-spectrophotometric assay is used. Silica gel-impregnated glass microfiber sheets (e.g. ITLC sheets of the Gelman Instrument Co., Ann Arbor, Mich.) are impregnated with silver nitrate, using 5% ethanolic silver nitrate and drying. Spots of 100 to 200 ,ug of the F'GA mixture are applied and developed in the solvent system 2,2.4-trimethy1pentanezethyl acetate: acetic acid: water (100:35:8:10, upper'phase). The sheet is dried and sprayed with Rhodamine 6G (Applied Science Co., State College, Pa) and viewed under ultraviolet light. The areas containing the cis and trans materials R, of PGA 0.6; R, of 5,6-trans-PGA 0.7) are marked, then excised and eluted with methanol 1.9 ml.) and potassium hydroxide solution (0.1 ml. of 45%). After incubation at 40 for 30 min., the respective solutions are centrifuged and analyzed spectrophotometrically at 278 nm.

Following the procedure of Preparation 6, 5,6-trans- 15,8-PGA is separated from 15B-PGA Preparation 7: PGE and 5,6-trans-PGE Following the procedure of Preparation 6, PGE is separated from 5,6-trans-PGE as follows. A solution of a mixture of PGE and 5,6-trans-PGE is charged to the column. Elution with 3A alcohol yields fractions which are combined according to their content of 5,6-trans- PGE (faster eluting) or PGE Assay for 5,6-trans- PGE or PGE is done by TLC as for the PGA -type compounds above. R, of POE- is 0.13; R, of 5,6-trans- PGE is 0.17. Combined fractions are concentrated, dried over sodium sulfate, and concentrated under reduced pressure to yield the title compounds.

Preparation 8: PGA l5-Acetate Methyl Ester, separation from 5,6-Trans-PGA IS-Acetate Methyl Ester.

A mixture of PGA; l5-acetate methyl ester and 5.6- trans-PGA IS-acetate methyl ester l 1.0 g., :15) is dissolved in 415 ml. of a solution of methanol-wateracetic acid (-5-04) and mercuric acetate (6.1 g.), and left standing at about 25 C. for 30 min. Water (250 ml.) is added and the mixture extracted twice with 700 ml. of Skellysolve B. The Skellysolve B phase is washed with ml. of 60% methanol, dried over sodium sulfate, and concentrated to an oil (4.35 g.) having a high content of 5,6-trans-IPGA 15-acetate methyl ester. The aqueous methanol phase is acidified with 32 ml. of 6 N. hydrochloric acid and the mixture is extracted with two portions of 700 ml. of Skellysolve B. The organic phase is dried over sodium sulfateand concentrated to an oil (5.53 g.) This last material is subjected to the same procedures again. using 350 m1. of the methanol-water-acetic acid and 4.6 g. of mercuric acetate. There is recovered from the work-up of the aqueous-methanol phase a fraction (3.92 g. )of the title compound containing only a small percentage of the 5,6-trans-PGA compound. i

Following the procedure of Preparation 8,5,6-translSB-PG A IS-acetate methyl ester is separated from 15B-PGA IS-acetate methyl ester.

1n the following examples, the above-described 5,6- trans-PG and 5,6-trans-l5B-PG compounds are subjected to the same transformations as the P0 and 15B- PG compounds disclosed herein and illustrated hereafter.

EXAMPLE 1 15B-PGA Methyl Ester A solution of 70% aqueous perchloric acid (50 drops) in 50 drops of water is added to a solution of 15B-PGA acetate methyl ester (2.0 g.) in 100 ml. of methanol. The mixture is stirred for 15 hours at 25 C. and then diluted with 80 ml. of water. The methanol is removed under reduced pressure. and the aqueous residue is extracted with ethyl acetate. The extract is washed successively with water and brine, dried with anhydrous sodium sulfate, and evaporated. The residue is chromatographed on 200 g. of silica gel. eluting with 2.5 l. of a gradient of 20-7072 ethyl acetate in Skellysolve B (a mixture of isomeric hexanes), collecting 100-ml. fractions. Fractions 15-19 are combined and evaporated to give 727 mg. of 1SB-PGA methyl ester.

EXAMPLE 2 FGA Methyl Ester A solution of 15,8-PGA methyl ester (250mg. in 20 ml. of anhydrous tetrahydrofuran is cooledto C. in an atmosphere of nitrogen. Tributylaminc 15 ml.) is added. and the mixture is stirred at 0 C. while adding methanesulfonyl chloride (1 ml.) dropwise. The mixture is stirred 30 minutes at 0 C. Then, 10 ml. of water is added, and the mixture is allowed to warm to 25 C. and is stirred for one hour. The tetrahydrofuran is evaporated under reduced pressure, and the aqueous residue is extracted with ethyl acetate. The extract is washed successively with one N hydrochloric acid, water, and brine, dried with anhydrous sodium sulfate, and evaporated. The residue is chromatographed on 30 g. of silica gel, eluting with 800 ml. of a gradient of 2070% ethyl acetate in Skellysolve B, collecting 25- ml. fractions. Fractions 14-16 are combined and evaporated to give 58 mg. of PGA methyl ester. Fractions 12 and 13 are combined to give 49 mg'. of the starting material, 15B-PGA methyl ester. 7

Following the procedure of Example 2, PGA methyl ester is transformed to a mixture of PGA and 15,8- PGA methyl esters, the two compounds being separated as in Example 2.

EXAMPLE 3 PGA l-formate and 15B-PGA 15-formate A solution of sodium carbonate (50 mg.) in 7.5ml. of anhydrous formic acid is added to PGA (0.25 g.). This mixture is stirred under nitrogen at 25 C. for 2 hrs. The reaction mixture is concentrated under reduced pressure, taken up in benzene, and again con V centrated under reduced pressure. The residue is chromatographed on acid-washed silica gel (e.g. Mallinckrod Silicar CC-4), eluting. with a gradient of 2575% ethyl acetate-Skellysolve B (isomeric hexane mixture) and collecting fractions. Those fractions shown by TLC to contain the respective l5-formate compound, separated from its isomer and free of startingmaterial and impurities, are combined and concentrated under reduced pressure to give the title compounds.

EXAMPLE 4 PGA and l5B-PGA PGA l5-formate (100 mg., Example 3) is dissolved in a mixture of ml. of methanol and 2.5 ml. of saturated aqueous sodium bicarbonate solution. The solution is stirred under nitrogen at 25 C. for 2.5 hrs. Then 5 ml. of water and 2 ml. of l N. hydrochloric acid are added, and the solution concentrated. The aqueous residue is adjusted to pH 2-3 and extracted three times with ethyl acetate. The combined extracts are washed with water, dried over sodium sulfate, and concentrated to yield PGA Similarly, hydrolysis of 15,8-PGA -formate (Example 3) yields lSB-FGA,

EXAMPLE 5 15B- PGA 10,1 l-Epoxide Acetate Methyl Ester Hydrogen peroxide (350 ml,; 30% aqueous) is added with stirring to a solution of ISB-PGA acetate methyl ester (265 g.) in 5000 ml. of methanol under a nitrogen atmosphere at C. Then, one N aqueous potassium hydroxide solution (50 ml.) is added gradually during one hour with stirring at 20 C. The mixture is stirred an additional 2 hours at 20 C. Then, one N hydrochloric acid ml.) is added, and the methanol is removed under reduced pressure at 35 C. The resiude is dissolved in 3000 ml. of ethyl acetate, and the solution is washed 3 times with 500-ml. portions of water. The combined water washes are extracted with 300 ml. of ethyl acetate. The two ethyl acetate solutions are combined, washed with brine, dried with anhydrous sodium sulfate and evaporated to give 2.75 g. of a mixture of the alpha and beta l0,11-epoxides ISB-PGA acetate methyl ester.

EXAMPLE 6 PGA 10,1 l-Epoxide Methyl Ester Hydrogen peroxide (0.3 ml., 30% aqueous) and one N aqueous sodium hydroxide (0.5 ml.) are added to a solution of PGA methyl ester (229 mg.) in 10 ml. of isopropyl alcohol at 0 C. After 2.5 hours at 0 C., 10 ml. of water and one ml. of one Nhydrochloric acid are added, and the isopropyl alcohol is removed under reduced pressure. The residue is extracted with ethyl acetate. The extract is washed successively with water and brine, dried with anhydrous sodium sulfate, and evaporated. The residue is chromatographed on 30 g. of silica gel, eluting with 800 ml; of a gradient of:2070% ethyl acetate in Skellysolve B, collecting 25-ml. fractions. Fractions 5-10 are combined and evaporated to give 136 mg. of a mixture of the alpha and beta 10,11- epoxides of PGA methyl ester. 1

EXAMPLE 7 PGA Acetate Methyl Ester a and B 10,11-Epoxides Refer to Chart A. i

A solution of PGA; l5-acetate methyl hydrochloric (1.954 g.) in 30 ml. of dimethoxyethane (DME) .is cooled to 55 C. under nitrogen, and 5.25 ml. of tbutyl hydroperoxide is added. Then, 5 ml. of 0.25 N. methanolic lithium hydroxide (prepared from the mono-hydrate is added over min. After about 46 hrs. an additional 2.5 ml.:of the base is added over 50 min. Finally, after about 23.5 hrs. the reaction is complete, as shown by TLC. The mixture'is acidified to pH 56 with l N hydrochlorici acid and is concentrated under reduced pressure. The residue is taken up in ethyl acetate, washed with brine, dried over sodium sulfate, and concentrated under reduced pressure. The product, 2.0 g., contains the title alpha and beta compounds in a ratio of 6: 1, respectively, as shown by gas chromatography.

Following the procedures of Example 7, but replacing the lithium hydroxide solution with methanolic magnesium methoxide (prepared from magnesium and anhydrous methanol), there is obtained a product containing the alpha and beta epoxides in a ratio of 4:1.

Following the procedures of Example 7, but replacing the DME witha mixture of toluene-DME (10:1) and replacing the lithium hydroxide with Triton B (benzyltrimethylammonium hydroxide) in methanol. there are obtained the alpha and beta epoxides in a ratio of 7.2:1

Following the procedures of Example 7, but replacing the DME with a mixture of toluene-DME 1:1 and holding the reaction temperature at 40 C., the product contains the alpha and beta epoxides in a ratio of 6.2:1.

Following the procedures of Example 7, but replacing the DME with tetrahydrofuran (THF) and replacing the lithium hydroxide solution with Triton B, there are obtained the alpha and beta epoxides in a ratio of 4.5:1.

EXAMPLE 8 lB-PGE l5-Acetate Methyl Ester and l IBJSB-PGE l5-Acetate Methyl Ester.

Granular aluminum metal (50 g.) is added to a solution of mercuric chloride (50 g.) in 2.1 of water. The mixture is swirled until hydrogen gas evolution starts to become vigorous (about 30 seconds). Then, most of the aqueous solution is decanted, and the rest is removed by rapid filtration. The amalgamated aluminum is washed rapidlyand successively with two 200-ml. portions of methanol and two ZOO-ml. portions of anhydrous diethyl ether. The amalgamated aluminum is then covered with anhydrous diethyl ether until used.

Methanol (250 ml.) and water (25 ml.) are added to a solution of a mixture of the alpha and beta 10,11- epoxides of l5B-PGA acetate methyl ester (275 g.) in 2500 ml. of diethyl ether. The mixture is cooled to C. and the amalgamated aluminum prepared as above filtered. The filter cake is washed thoroughly with dichloromethane, and the combined filtrate and washings are evaporated at C. under reduced pressure to give a mixture (247 g.) of ISB-PGE l5-acetate methyl ester and l 1B,15,-2-PGE l5-acetate methyl ester..

Part of this mixture (210 g.) is chromatographed on kg. of silica gel wet-packed with 60 l. of 25% ethyl acetate in Skellysolve B (6-inch diameter column), eluting successively with 60-1. portions of 25%, 30%, and ethyl acetate in Skellysolve B, collecting 4-l. fractions. Fractions 71-76 are combined and evaporated to give 27 g. of l 1,8,15,8- PGE l5-acetate methyl ester. Fractions 81-98 are combined and evaporated to give 1 15 g. of ISB-PGE 15-acetate methyl ester.

EXAMPLE 9 l5B-PGE 15-Acetate Methyl Ester and l lB,l5B-PGE l5-Acetate Methyl Ester.

Anhydrous sodium acetate (0.5 g.) and zinc dust (500 mg.) are added to a solution of a mixture of the alpha and beta'epoxides of 15, 3-PGA acetate methyl ester, prepared as in Example 5, in 5 ml. of acetic acid. This mixture is stirred at 25 C. in atmosphere of nitrogen and cooled to'about 15 C. One-half ml. of a solution of chromium (lIl) chloride hexahydrate (300 mg. in l ml. of water is added, and the mixture is stirred at 0 C. for 3 hours. The mixture is then diluted with ethyl acetate, and the solution is washed successively with four portions of water, one N hydrochloric acid, sodium bicarbonate solution, and brine, dried with anhydrous sodium sulfate, and evaporated. The residue is chromatographed on 20 g. of silica gel, eluting with 600 ml. ofa gradient of 20-75% ethyl acetate in Skellysolve B, collecting 25-ml. fractions. Fractions l0 and l l are combined to give 11 BJSB-lPGE l5-acetate methyl esterIFractions l3 and 14 are combined to give 15,8- PGEg IS-acetate methyl ester.

. Example 10 PGE Methyl Ester and 1 l/S-PGE Methyl Ester.

Freshly prepared chromium (ll) acetate (450 mg, argon atmosphere; Inorganic Syntheses, 8, is added to a solution of 136 mg. of epoxides (Example 6) in a mixture of 3 ml. of acetic acid and one ml. of water in an atmosphere of argon at 0C. The mixture is stirred at 5 C. under argon for 18 hours. Ice is then added to the mixture, and that mixture is extracted with ethyl acetate. The extract is washed successively with water, one N hydrochloric acid, sodium bicarbonate solution, and brine, dried with anhydrous sodium sulfate, and evaporated. The residue is chromatographed on silica gel (20 g.), eluting with 600 mli ofa gradient of 20-100% ethyl acetate in Skellysolve B, collecting 20 ml. fractions. Fractions 19-22 are combined and evaporated to give 27 mg. of llB-PGE methyl ester. Fractions 24-27 are combined and evaporated to give 5 mg. of POE-,- methyl ester.

EXAMPLE 1-1 PGEZ and ins-P615 with stirring. After 1 hour of stirring at 20. C., one ml.

of one N hydrochloric acid is added, and the mixture is evaporated under reduced pressure. The residue is dissolved in ethyl acetate, and the resulting solution is washed successively with water and brine, dried with anhydrous sodium sulfate, and. evaporated. The residue is treated with amalgamated aluminum as described in Example 8, using 2.5 ml. diethyl ether, 0.25 ml. methanol, and 003ml. water, the amalgamated aluminum being added in 2 portions. When the reduction is complete, ethyl acetate and one N hydrochloric acid are added to the reaction mixture and the mixture is separated in a separatory funnel. The ethyl acetate layer is washed successively with onell hydrochloric acid, water, and brine, dried with anhydrous sodium sulfate, and evaporated. The residue is subjected to preparative thin layer chromatography to give PGEZ and 11B- PGE in the ratio of 1 :2.

7 EXAMPLE 12 ISB-PGE and l lfl,l5B-PGE Following the procedure of Example 1 l, 15,8-PGA is transformed to l5B-PGE and 1 lB,l5,8-PGE those being obtained in the ratio l:l.

EXAMPLE l3 .PGE and l lB-PGE Hexamethyldisilizane (1 ml.) and trimethylchlorosilane (0.2 ml.) are added with stirring to a solution of PGA (250 mg.) in 4 ml. of tetrahydrofuran at 0 C. under nitrogen. This mixture is maintained at 5 C. for 15 hours. The mixture is then evaporated under reduced pressure. Toluene is added and evaporated twice. Then the residue is dissolved in 6 ml. of metha- 

1. A COMPOUND OF THE FORMULA:
 2. A compound according to claim 1 wherein G is
 3. A compound according to claim 1 wherein G is
 4. A compound according to claim 2 wherein R2 is methyl and R5 is acetyl.
 5. A compound according to claim 2 wherein R2 is methyl and R5 is -Si-(A)3.
 6. A compound according to claim 5 wherein A is methyl.
 7. A compound according to claim 3 wherein R2 is hydrogen and R5 is -Si-(A)3.
 8. A compound according to claim 7 wherein A is methyl.
 9. A compound according to claim 3 wherein R2 is methyl and R5 is -Si-(A)3.
 10. A compound according to claim 9 wherein A is methyl. 